Nucleoid structuring proteins play important roles in the transcriptional silencing of many bacterial genes. Transcription factors that alleviate this silencing are central to many bacterial processes, including virulence gene expression, biofilm formation and bacterial adaptation to different environments, all of which play important roles in bacterial virulence. A better understanding of transcriptional silencing and anti-silencing mechanisms is warranted. The icsP gene, which is carried by the Shigella virulence plasmid, is transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by the transcription factor VirB. Nine putative VirB-binding sites lie upstream of the icsP gene. Mutagenesis of the two most distal sites, located over 1 kb upstream of the icsP transcription start site, completely destroys VirB-dependent regulation of this promoter. It is highly unusual for bacterial transcription factors to affect transcription from such remote sites. This observation raises several questions that are central to virulence gene expression in Shigella and other pathogens. How does VirB function to alleviate H-NS dependent repression of the icsP promoter from such distances? Does VirB function from remote sites to regulate the expression of other Shigella virulence genes? Can transcription factors found in other bacteria function from remote sites to alleviate transcriptional repression by nucleoid structuring proteins? If so, how widespread is this phenomenon? These questions will frame our future studies. The work proposed here will determine how VirB regulates the activity of the icsP promoter. In specific aim one, the seven remaining VirB-binding sites will be destroyed in the presence of the two distal sites and the activity of each construct will be measured in vivo. To assess which of the nine VirB-binding sites are likely to initiate VirB binding to the icsP promoter electrophoretic mobility shift assays will be used. In specific aim two, the role that an area of intrinsically curved DNA plays in the H-NS and VirB-dependent regulation of the icsP promoter will be tested. Single pairs of base substitutions will be introduced into the icsP promoter DNA to diminish its curvature. The effect these substitutions have on the curvature of the DNA and the regulation of the icsP promoter by VirB and H-NS will be measured. The innovation of this project lies in the use of icsP promoter to study two phenomena that are central to the fields of bacterial pathogenesis and bacterial gene regulation i) how genes are repressed by nucleoid structuring proteins and derepressed by bacterial transcription factors, and ii) how bacterial transcription factors alleviate this repression from remotely located binding sites. Since nucleoid structuring proteins regulate the expression of many genes in both gram negative and gram positive pathogens, these studies will have broad significance. PUBLIC HEALTH RELEVANCE: The proposed studies will be conducted at the University of Nevada, Las Vegas by undergraduate and graduate students. The project will provide these students research opportunities in the field of bacterial gene expression and transcriptional regulation, an area that is central to bacterial pathogenesis and health related research.